Structural Analysis and Development of Notum Fragment Screening Hits

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer’s disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments’ diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.


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Page S3 Figure S1. Schematic illustration of the overall procedure for the X-ray based fragment screen, lead identification and development.
Page S6 Procedures for the purchase or resynthesis of fragment hits 1-58.
Page S56 Figure S4. UHPLC analytical data for advanced lead 7d.
S3 Figure S1. Schematic illustration of the overall procedure for the X-ray based fragment screen, lead identification and development. Table S1. Notum inhibition SAR for additional pyrrazoles 16, benzylamines 28 and benzimidazoles 15.
Additional pyrrazoles 16b-f, benzylamines 28b-j and benzimidazoles 15b-m were prepared by the general methods as described and Notum inhibition data presented here for a more complete analysis of the SARs. The most active member is in bold text. S7 bright orange solution, acetyl chloride (0.05 mL, 0.74 mmol) was then added and the reaction allowed to warm to room temperature and stirred for 3 hours. After this time further acetyl chloride (0.05 mL) was added and the reaction was left to stir overnight at room temperature under nitrogen.
After this time the reaction was diluted with water and basified using NaOH 1N solution. The organics were extracted with dichloromethane three times. The organics were combined, dried over anhydrous

4-Methoxy-6-phenyl-pyrimidin-2-amine (46)
A solution of 2-amino-6-phenylpyrimidin-4-ol (400 mg, 2.14 mmol) in N,N-dimethylformamide (6 mL) was cooled to 0 o C and for 0.5 hours before sodium hydride (94 mg, 2.35 mmol) was added. The mixture was stirred for 15 min, before iodomethane (0.13 mL, 2.14 mmol) was then slowly added and stirred for a further 1 hour. After this time the mixture was warmed to room temperature and quenched by pouring onto sat. aq. NaHCO 3 (10 mL). The reaction mixture was diluted with dichloromethane (10 mL) and the organics were then separated, dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.